Paclitaxel is a well known chemotherapeutic drug for treatment of various metastatic cancers. It has been approved by the Food and Drag Administration (FDA) for the treatment of ovarian and breast cancers and is currently in clinical trials for the treatment of lung and colon cancers.
The compound is a natural product primarily extracted from the bark of the Pacific Yew tree, Taxus brevifolia, and is also found in T. baccata, T. walichiana and T. yunnanensis and other biomass extracts from plant materials including T. hicksii, T. densiformis, T. gem, T. wardii, T. cuspidata, T. capitata, T. brownii, and T. dark green spreader, which contain a mixture of taxane-type molecules. Paclitaxel is also available from cultured plant cells and fungi. The compound is commercially available in reagent grade, for example, from the Aldrich Chemical Co., product No. 41,701-7, Sigma Chemical Company, product Nos. T 7402 and T 1912, depending on the source it was derived from, Fluka Chemie AG. product No. 86346 and ICN Biomedicals product No. 193532.
The concentration of paclitaxel in various raw materials is typically low, for example, on the order of between 0.0004-0.08% (w/w) in the bark of the Pacific Yew. Such low concentrations render the extraction and purification of the compound to clinical standards from raw materials very challenging, and heretofore impractical on a commercial level.
Presently, several processes for the extraction and purification of paclitaxel are known. Wani et al., J. Am. Chem. Soc. 93,9:2325-2327 (1971), describes extraction of T. brevifolia stem bark with ethanol, which is then concentrated and extracted with chloroform and water, and wherein the paclitaxel is found in the chloroform phase. The paclitaxel is further purified by column chromatography over florisil, sephadex, and silica gel columns.
A National Cancer Institute (NCI) method (1983) is based on extraction of T. brevifolia stem bark with methanol followed by methylene chloride extraction. The methylene chloride extract is dried and then dissolved in acetone followed by precipitation of impurities with n-hexane. The soluble fraction is further purified by column chromatography.
Both the Wani et al. and NCI procedures, however, are not very efficient or commercially practical as they result in very low yields on the order of about 0.02% or less. This is due to the presence of other taxanes, such as paclitaxel's close analog cephalomannine, having similar structures and very close physical properties to that of paclitaxel. See FIG. 1 which illustrates the chemical structures of paclitaxel and cephalomannine.
In a process developed by Potier et al., J. Nat. Prod., 47,1:131-137 (1984), the precipitation step in the NCI process is substituted for a step employing a solvent pair extraction method, i.e. using successive extractions with progressively increasing polar solvents. After further steps of chromatography over alumina and silica columns, the paclitaxel is concentrated as a mixture of paclitaxel and cephalomannine. The paclitaxel is then separated from cephalomannine by HPLC, with a yield of paclitaxel considerably higher than obtained by either of the Wani et al. or NCI methods.
However, the Potier et al. method, in similar manner as the Wani et al. and NCI methods, suffers from the major drawback of requiring the separation of multiple taxanes with similar separation parameters in a final concentrate by the use of multiple conventional chromatographic separations to obtain a purified paclitaxel product. As large scale commercial processing of paclitaxel employing multiple conventional chromatographic separations to provide clinically acceptable pure paclitaxel would be necessitated by these methods, such are commercially impractical because of the large expense associated with such multiple chromatographic separations.
Multiple separations are necessitated for the most part by the similarity in both structure and properties of paclitaxel with cephalomannine. As shown in FIG. 1, the only difference in their structures is that the amino group in the side chain in paclitaxel is acylated with benzoic acid, and in cephalomannine the side chain amino group is acylated with tiglic acid containing a double bond.
Methods other than chromatographic separation of paclitaxel from cephalomannine are known, such as chemical modifications of the side chain double bond in cephalomannine. For example, Kingston, et al., J. Nat. Prod., 55: No. 2, 259-261 (1992) describes the catalytic oxidation of the cephalomannine side chain double bond in the presence of OsO.sub.4 to obtain a diol, which is then separated from paclitaxel by chromatographic procedures and recrystallizations. There are problems with this method in the use of unpurified taxane mixtures since the oxidation catalyzed by OsO.sub.4 is not amenable to crude extracts due to low selectivity for the side chain double bond of cephalomannine, which if could be used, would significantly reduce the cost of the extraction and purification process. Additionally, using OsO.sub.4 in the manufacture of pharmaceuticals is not desirable due to the severe toxicity of the compound.
In U.S. Pat. Nos. 5,334,732 and 5,336,684 to Murray, et al., oxidation of the cephalomannine side chain by ozone is described. These methods are also undesirable in that the use of ozone in an oxidation process with crude extracts produces many unwanted reactions with paclitaxel; oxidation by ozonolysis is strong and not selective in compounds with many functional groups such as paclitaxel and cephalomannine, and may cause unwanted oxidations of other functional groups, such as aldehydes, ketones, amines, etc. in the paclitaxel molecule or of the double bond found on the inside of the taxane ting of either paclitaxel or cephalomannine. There is also the expensive requirement of an ozone generator.
Thus, the isolation and purification of paclitaxel from a raw biomass-containing complex mixture of taxanes or, at the other extreme, from more purified mixtures containing primarily paclitaxel and cephalomannine is currently limited to the aforesaid non-economical chromatographic separation techniques and/or to non-selective oxidation methods, thereby presenting a serious and unfulfilled need for an economically practicable method for separating the valuable anti-tumor compound paclitaxel from its close analog cephalomannine, as well as other closely related taxanes.
It is therefore an object of this invention to provide a simpler and more cost effective method than currently available methods for the economical isolation and purification of the important chemotherapeutic compound paclitaxel.